Polysaccharides are essential biopolymers performing diverse biological functions, ranging from energy storage to osmoregulation and cell wall formation. Extracellular polysaccharides, including cellulose, chitin, and alginate, are synthesized inside the cell from nucleotide-activated sugars and are transported across the cell membrane during their synthesis. This remarkable task is performed by membrane-integrated glycosyltransferases (GT) that couple polymer elongation with translocation (1, 2). Cellulose is the most abundant biopolymer on earth primarily formed by vascular plants, but also by some bacteria. Bacterial extracellular polysaccharides, such as cellulose and alginate, are an important component of biofilms, which are multi-cellular, usually sessile, aggregates of bacteria. Biofilms exhibit a greater resistance to antimicrobial treatments compared to isolated bacteria and thus are a particular concern to human health.
Cellulose is a linear polymer of glucose molecules linked via β-1,4 glycosidic linkages (3, 4) and is primarily formed by vascular plants, but also by some algae, protists, and bacteria (4-6). Cellulose is synthesized by cellulose synthase (CesA), a family 2 member of GT (7) that processively polymerizes UDP-activated glucose via an evolutionarily conserved mechanism (2). CesAs contain eight predicted transmembrane (TM) segments and at least one extended intracellular domain adopting a GT-A fold (2, 8). The intracellular GT-A domain is responsible for donor and acceptor sugar binding as well as for catalyzing the GT reaction and the membrane-embedded part forms a TM pore in close juxtaposition with the catalytic site, thereby allowing translocation of the nascent polysaccharide (2).
While most eukaryotic CesAs are believed to form supra-molecular complexes that organize the secreted glucans into cable-like structures, i.e. the cellulose microfibrils (9), many Gram-negative bacteria synthesize cellulose as a biofilm component (10, 11). Biofilm formation is stimulated by the bacterial messenger cyclic-di-GMP (c-di-GMP) (12), which affects a diverse group of enzymes via interaction with either covalently or non-covalently attached c-di-GMP-binding domains, such as PilZ (13-15).
Bacterial cellulose synthase (Bcs) is a multi-component protein complex encoded in an operon containing at least 3 genes, bcsA, -B and -C (16, 17). While BcsA is the catalytic subunit that synthesizes cellulose and forms the TM pore across the inner membrane, BcsB is a large periplasmic protein that is anchored to the inner membrane via a single C-terminal TM helix. BcsB may guide the polymer across the periplasm towards the outer membrane via two carbohydrate-binding domains (CBD) (2). BcsA and BcsB are fused into a single polypeptide chain in some species (18). BcsC is predicted to form a β-barrel in the outer membrane, preceded by a large periplasmic domain containing tetratricopeptide repeats likely involved in complex assembly (16). Most cellulose synthase operons also code for a periplasmic cellulase, BcsZ, whose biological function is unknown, yet it appears to enhance cellulose production in vivo (19, 20). While most biofilm-forming bacteria likely produce amorphous cellulose that is embedded in a 3-dimensional matrix of polysaccharides, proteinaceous fibers and nucleic acids (21), some bacteria produce cellulose microfibrils resembling those synthesized by eukaryotic cells (22). In such bacteria, CesA complexes are linearly arranged along the cell axis and the CesA operons encode at least one additional subunit, BcsD, that might facilitate the linear organization of the synthases (18).
Despite the numerous studies available on a large number of pro- and eukaryotic model systems, revealing the mechanism of cellulose synthesis and translocation has been hampered by difficulties in reconstituting functional cellulose synthases in a purified system, either from eukaryotic or prokaryotic enzymes (23-26). To date, cellulose biosynthetic activities have only been recovered from detergent extracts of native membranes (24-26).
There is a long felt need in the art for compositions and methods useful making cellulose in an acellular manner. The present invention satisfies these needs.